Development of mPMab-1,a Mouse–Rat Chimeric Antibody Against Mouse Podoplanin

Introduction

Podoplanin (PDPN) is expressed in many normal tissues such as podocytes of the kidney, lymphatic endothelial cells of many tissues, and type I alveolar cells of the lung.⁽¹⁾ Several anti-mouse PDPN monoclonal antibodies (mAbs) such as clone 8.1.1 or PMab-1 are commercially available.^(1,2) However, 8.1.1 is produced using hamsters and PMab-1 is produced using rats because it is difficult to develop anti-mouse PDPN mAbs using mice. PMab-1 is produced against the platelet aggregation-stimulating domain of mouse PDPN⁽¹⁾; therefore, PMab-1 neutralizes the interaction between PDPN and C-type lectin-like receptor 2 (CLEC-2). Administration of PMab-1 reduces lymphangiogenesis in the corneal suture and ear wound healing models.⁽³⁾ PMab-1 also suppresses the infiltration of thioglycollate-induced macrophages at the site of wound healing. Furthermore, administration of PMab-1 leads to a significant suppression of the rejection reaction in the corneal transplantation model, suggesting that PDPN is a novel therapeutic target for suppressing lymphangiogenesis and inflammation.

In this study, we produce a mouse–rat chimeric antibody, mPMab-1, which is derived from PMab-1.

Materials and Methods

Results and Discussion

We previously established PMab-1 of rat IgG_2a as a specific mAb against mouse PDPN. PMab-1 neutralizes the interaction between PDPN and CLEC-2. PMab-1 is also very sensitive in the immunohistochemical analysis. In contrast, rat mAbs seem to be unfavorable for pathologists because anti-rat IgG is not included as secondary antibody in commercially available kits for immunohistochemical analysis such as Envision+ (Dako). Nevertheless, mouse anti-mouse PDPN antibodies have not been reported. Therefore, we herein produced a mouse–rat chimeric antibody, mPMab-1, which was derived from PMab-1.

We first checked the reactivity of mPMab-1 in flow cytometry. The reactivity of mPMab-1 against CHO/mPDPN cells is compatible to that of PMab-1 (Fig. 1). To investigate whether mPMab-1 is useful in immunohistochemical analysis using commercially available kits, we next compared the reactivity between PMab-1 and mPMab-1 against normal mouse tissues. mPMab-1 reacted with renal podocytes (Fig. 2A, B) in the same way as PMab-1 (Fig. 2C, D). Importantly, the reactivity of mPMab-1 is higher than that of PMab-1. Without primary antibodies, slight unspecific staining was observed in red blood cells of the renal glomerulus (Supplementary Fig. S1A, B). Both mPMab-1 (Fig. 3A, B) and PMab-1 (Fig. 3C, D) also stained lymphatic endothelial cells and fibroblasts of the colon. The signal of mPMab-1 is also higher compared with PMab-1. Furthermore, both mPMab-1 (Fig. 4A, B) and PMab-1 (Fig. 4C, D) stained type I alveolar cells of the lung, indicating that mPMab-1 is very useful for immunohistochemical analysis using commercially available immunohistochemical kits.

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FIG. 1.

Flow cytometric analysis of anti-mouse PDPN mAbs. Cells were treated with PMab-1 and mPMab-1 (1 μg/mL) or control PBS (black) for 30 minutes at 4°C, followed by treatment with anti-rat or mouse IgG Oregon Green. Fluorescence data were collected using a Cell Analyzer EC800. mAb, monoclonal antibody; PBS, phosphate-buffered saline; PDPN, podoplanin.

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FIG. 2.

An immunohistochemical analysis using anti-mouse PDPN mAbs to detect PDPN expression in the kidney. Sections were incubated with mPMab-1 (A, B) or PMab-1 (C, D) and made to react with the Envision+ kit. Color was developed using DAB, and samples were then counterstained with hematoxylin. Sections were also stained with hematoxylin and eosin (E, F). Scale bar = 100 μm.

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FIG. 3.

An immunohistochemical analysis using anti-mouse PDPN mAbs to detect PDPN expression in the colon. Sections were incubated with mPMab-1 (A, B) or PMab-1 (C, D) and made to react with the Envision+ kit. Color was developed using DAB, and samples were then counterstained with hematoxylin. Sections were also stained with hematoxylin and eosin (E, F). Scale bar = 100 μm.

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FIG. 4.

An immunohistochemical analysis using anti-mouse PDPN mAbs to detect PDPN expression in the lung. Sections were incubated with mPMab-1 (A, B) or PMab-1 (C, D) and made to react with the Envision+ kit. Color was developed using DAB, and samples were then counterstained with hematoxylin. Sections were also stained with hematoxylin and eosin (E, F). Scale bar = 100 μm.

In this study, PMab-1 was also shown to be useful in immunohistochemical analysis, although anti-mouse secondary antibodies are included in immunohistochemical kits. Anti-mouse secondary antibodies usually cross-react with rat antibodies; cross-reaction seems to possess a lower reliability for pathologists and scientists. mPMab-1 could be used as mouse IgG and its reactivity is higher than that of PMab-1. This conversion strategy from rat mAb to mouse antibody could be applicable to other mAbs.